Innate immune cells constantly evaluate host mucosal surfaces and peripheral tissues for signs of infection or injury. The host must find a balance between tolerance of beneficial microorganisms and minor non-pathological microbial encounter vs. the development of a robust immune response to more serious infections. Emerging evidence suggests that this decision is made by the cell based on the strength and combination of signals it receives from its engagement with microorganisms and endogenous stimuli. These signals are sensed primarily by various classes of pattern recognition receptors (PRR), and while there has been remarkable progress in characterizing the individual signaling pathways induced by these receptors, relatively few studies have addressed how immune cells integrate combined PRR inputs and the combination of these signals with others arising from soluble host derived substances such as cytokines, lipids, and metabolites. We have previously analyzed the macrophage response to single and pairwise combinations of toll-like receptor (TLR) ligands, identifying characteristics of signaling pathway synergy and antagonism in signaling and cytokine outputs (Lin et al (2017) Cell Syst. 5: 25; Gottchalk et al (2016) Cell Syst. 2: 378). These studies emphasized the importance of feedback control in regulation of signaling flux, and in FY18 we have further investigated this control phenomenon on two fronts. Firstly, our prior screening work suggested a negative regulatory role for the protein TANK in synergy between the Myd88 and TRIF branches of the TLR signaling response (Lin et al (2017) Cell Syst. 5: 25). Using primary macrophages from TANK-deficient mice, we now demonstrate that this negative regulatory function acts upstream of the primary MAPK and NF-kB signaling pathways. We are uncovering mechanistic aspects of this regulation and we also demonstrate a critical physiological role for this negative regulation in characterizing a pre-disposition to auto-inflammation in aging TANK-deficient mice. Secondly, in collaboration with the Lymphocyte Biology Section of the LISB, a rigorous analysis of macrophages exposed to a matrix of increasing concentrations of paired TLR ligands has identified an acute negative feedback phenomenon that is selectively engaged during gram-negative bacterial infection. This anti-inflammatory feedback control is dependent on specific negative regulatory genes that are induced by type-I IFN, and does not involve the established IL-10-dependent feedback loop. A manuscript describing this work has been submitted for publication. To further address how the TLR signaling network might mediate responses specific to combined TLR stimuli, we have investigated the localization dynamics of proximal TLR pathway components in response to single vs. combined ligands. In this context, we previously identified an IRAK1-containing complex that directly links multi-TLR signaling to inflammasome activation. IRAK1 containing bodies, that were distinct from myddosomes and trifosomes, were formed on co-stimulation of TLR4 and TLR1/2 or on bacterial infection. We found these complexes simultaneously recruited the inflammasome adaptor ASC, facilitating dual-TLR ligand-primed inflammasome activation that was diminished in Irak1-deficient macrophages. In FY18, we gained additional insight into the function of IRAK1 as a non-transcriptional priming effector of the inflammasome. In a Yersinia pseudotuberculosis infection model where IL-1 responses are required for effective host defense, we also observe increased susceptibility and bacterial burden in Irak1-/- mice, suggesting a critical role for IRAK1-containing complexes in shaping the immune response to a multi-PAMP pathogen. This work will be submitted for publication in the near future. Further insight to the transcriptional programs activated by the Myd88 and TRIF-dependent signaling branches within the TLR pathways has arisen from our study of specific hits identified in our genome-wide screens of the LPS response in human macrophages (project AI001106). Activation of the TLR4 signaling pathway by LPS leads to induction of both inflammatory and interferon-stimulated genes, however, the mechanisms through which these coordinately activated transcriptional programs are balanced to promote an optimal innate immune response remain poorly understood. In our genome-wide siRNA screen of the LPS-induced TNF-alpha response, we identified the interferon-stimulated protein IFIT1 as a negative regulator of the inflammatory gene program. Transcriptional profiling further identified an unexpected positive regulatory role for IFIT1 in type I interferon expression, implicating IFIT1 as a reciprocal modulator of different LPS-induced gene classes. In FY18, we have confirmed that these effects are mediated by a specific nuclear pool of IFIT1 through modulation of a Sin3A-HDAC2 transcriptional regulatory complex at LPS-induced gene loci. Beyond the well-studied role of cytosolic IFIT1 in restricting viral replication, our data demonstrate an unappreciated function for nuclear IFIT1 in differential transcriptional regulation of separate branches of the LPS-induced gene program. This study was recently accepted for publication (John et al (2018) Cell Reports. In press). The above discovery of the regulatory role for IFIT1 on interferon-stimulated genes (ISGs) in the LPS response suggests an important role for this gene program in the host response to Gram-negative bacterial infection. To investigate this further, we have used the Gram-negative bacteria Burkholderia cenocepacia, which replicates in the host cell cytoplasm, to uncover a negative relationship between type I IFN signaling and the replicative potential of invading bacteria. Firstly, cells infected with these bacteria produce significant levels of IFN beta as well as the products of IFN-stimulated genes (ISGs). Cells pre-treated with IFN beta are less permissive to bacterial replication, while cells from mice lacking the type I IFN receptor (Ifnar1) have increased levels of bacterial replication, and higher amounts of cell death, compared to wild-type cells. Interestingly, this phenotype is type I IFN-specific, as pre-treatment with IFN-gamma has no effect on bacterial replication. In contrast, these phenotypes are reversed in cells infected with Salmonella, which replicates within in modified vacuole. This suggests that different IFN classes (and induced ISGs), may confer host protection against microbes replicating in different cellular niches. In FY18 we have conducted a broad transcriptomic profiling of bacterially infected WT and Ifnar1-deficient cells to identify the ISGs induced by different bacterial stimuli. This data is directing a pooled CRISPR screening strategy to identify which bacteria-induced ISGs are most important for protection of macrophages from intracellular bacterial replication. In FY18 we have also initiated studies to investigate the cell signaling pre-requisites that link the priming and triggering events that control inflammasome activation and release of the IL-1/IL-18 family of cytokines. This pathway is frequently dysregulated in the inflammatory and metabolic diseases that place a substantial burden on global human health, and inflammasome regulation is closely linked to TLR activation during its priming phase. Moreover, several of our projects suggest unappreciated roles for TLR pathway components in the inflammasome response, independent of their role in transcriptional induction of inflammasome genes. We are developing a number of strategies and signaling reporter assays to determine the influence of cell signaling pathways and processes (many of them strongly influenced by cellular metabolic state) to the inflammasome response. This line of enquiry is ongoing.